AIM: To investigate the effects of clozapine on apoptosis of
pancreatic β cells in male C57BL/6 mice.METHODS: The study was
carried out in the central laboratory of the First Affiliated Hospital of Nanjing Medical University from March to September 2004. Sixty-three male C57BL/6 mice of clean grade weighed 18 to 22 g were provided by Shanghai laboratory animal center certification number: SCXK (Shanghai) 2003-0003 . The mice were randomly divided into blank group (n=21), clozapine 4 mg/kg group (n=21) and clozapine 20 mg/kg group (n=21), and there were insignificant differences in the body mass and basic blood glucose among the groups (P > 0.05). The animals were housed (7 mice per cage) with free access to food and water in a temperature-regulated room in which the relative humidity was 45% with a 12-hour light/dark cycle (lights on 7:00-19:00). The mice were randomly divided into three groups, which administrated with distilled water, clozapine 4 mg/kg group, clozapine 20 mg/kg group, respectively. The weight and basic blood sugar were not different between the three groups. ①Model establishment: Clozapine was completely grinded and diluted with distilled water to prepare suspensions of 2 and 0.4 g/L respectively, which should be shaken to be even completely. The mice in the clozapine 4 mg/kg group were treated with gastric perfusion of 0.2 mL clozapine of 0.4 g/L), those in the clozapine 20 mg/kg group were treated with gastric perfusion of 0.2 mL clozapine of 2 g/L, and those in the blank group were given gastric perfusion of distilled water of the same dosage. According to the different time of attack, each group was subdivided into 3 groups with 7 mice in each: super-acute group were given gastric perfusion once, the acute group were treated with gastric perfusion once a day for 7 times, and the chronic group were treated with gastric perfusion once a day for 28 times. ② Sample treatment: Mice were fasted for 12 hours (8 Pm-8 Am), and they were killed by means of dislocation of cervical vertebra after blood collection by excising eyeballs at 3 hours after gastric perfusion in the super-acute group and 3 hours after the last gastric perfusion in the acute group (1 week) and chronic group (4 weeks), and then the pancreas was fixated in neutral formalin with the volume fraction of 0.1 for 24 hours and made into paraffin section with the thickness of 4 μm. Insulitis was observed under light microscope by hematoxylin-eosin (HE) staining. The apoptotic cells were marked by in situ nick end-labeling (TUNEL) and β cells were labeled by streptavidin-biotin-peroxidase complex (SABC), and the
apoptosis of βcells was counted.RESULTS: All the 63 animals were involved in the analysis of results. ① HE staining results: The edges of the
pancreatic islets were clear and the islets cells showed proportional distribution in the blank control group, clozapine 4 mg/kg group and clozapine 20 mg/kg group. There were no infiltration of lymph cells in the islets of pancreas. ② SABC results: Exterior and interior secretory portions were distinct. The β cells whose cytoplasm was buffy stained positively with insulin and there were no pancreatic apoptotic β cells in the blank control group, clozapine 4 mg/kg group and clozapine 20 mg/kg group.CONCLUSION: The effects of clozapine on apoptosis of pancreatic β cells in the male mice may be not conspicuous. Clozapine may not induce β cells apoptosis.